RNAi-related inhibition of TNFα signaling pathway for treatment of glaucoma

ABSTRACT

RNA interference is provided for inhibition of tumor necrosis factor α (TNFα) by silencing TNFα cell surface receptor TNF receptor-1 (TNFR1) mRNA expression, or by silencing TNFα converting enzyme (TACE/ADAM17) mRNA expression. Silencing such TNFα targets, in particular, is useful for treating patients having a TNFα-related condition or at risk of developing a TNFα-related condition such as the ocular conditions associated with elevated intraocular pressure (IOP), including glaucoma and ocular hypertension.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 12/184,403 filed Aug. 1, 2008; which claims priority under 35U.S.C. §119 to U.S. Provisional Patent Application No. 60/953,809 filedAug. 3, 2007, the entire contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to the field of interfering RNAcompositions for silencing tumor necrosis factor α (TNFα) by silencingthe TNFα cell surface receptor TNF receptor-1 (TNFR1) mRNA, or the TNFαconverting enzyme (TACE/ADAM17) mRNA. Silencing such TNFα targets isuseful for treatment of patients having a TNFα-related condition or atrisk of developing such a condition, wherein the condition is an ocularcondition associated with elevated intraocular pressure (IOP).

BACKGROUND OF THE INVENTION

Glaucoma is a heterogeneous group of optic neuropathies that sharecertain clinical features. The loss of vision in glaucoma is due to theselective death of retinal ganglion cells in the neural retina that isclinically diagnosed by characteristic changes in the visual field,nerve fiber layer defects, and a progressive cupping of the optic nervehead (ONH). One of the main risk factors for the development of glaucomais the presence of ocular hypertension (elevated intraocular pressure,IOP). An adequate intraocular pressure is needed to maintain the shapeof the eye and to provide a pressure gradient to allow for the flow ofaqueous humor to the avascular cornea and lens. IOP levels may also beinvolved in the pathogenesis of normal tension glaucoma (NTG), asevidenced by patients benefiting from IOP lowering medications. Onceadjustments for central corneal thickness are made to IOP readings inNTG patients, many of these patients may be found to be ocularhypertensive.

The elevated IOP associated with glaucoma is due to elevated aqueoushumor outflow resistance in the trabecular meshwork (TM), a smallspecialized tissue located in the iris-corneal angle of the ocularanterior chamber. Glaucomatous changes to the TM include a loss in TMcells and the deposition and accumulation of extracellular debrisincluding proteinaceous plaque-like material. In addition, there arealso changes that occur in the glaucomatous ONH. In glaucomatous eyes,there are morphological and mobility changes in ONH glial cells. Inresponse to elevated IOP and/or transient ischemic insults, there is achange in the composition of the ONH extracellular matrix andalterations in the glial cell and retinal ganglion cell axonmorphologies.

Primary glaucomas result from disturbances in the flow of intraocularfluid that has an anatomical or physiological basis. Primary open angleglaucoma (POAG), also known as chronic or simple glaucoma, representsninety percent of all primary glaucomas. POAG is characterized by thedegeneration of the trabecular meshwork, resulting in abnormally highresistance to fluid drainage from the eye. A consequence of suchresistance is an increase in the IOP that is required to drive the fluidnormally produced by the eye across the increased resistance.

Histopathologic studies of the glaucomatous optic nerve head in POAGreveal astroglial activation and tissue remodeling, which accompaniesneuronal damage. As a part of tissue remodeling, backward bowing anddisorganization of the laminar cribriform plates are commoncharacteristics of glaucomatous eyes with either normal or high IOP.These histologic changes are accompanied by the upregulation ofextracellular matrix components including collagen and proteoglycan, andadhesion molecules by optic nerve head astrocytes in glaucomatous eyes.The astroglial activation seen in glaucomatous optic nerve heads likelyrepresents an attempt to limit the extent of the injury and promote thetissue repair process. However, despite the astroglial activation, thereis limited deposition of extracellular matrix in glaucomatous opticnerve atrophy, which does not retain characteristics of scar tissueformation. This suggests that there are diverse cellular responses tothe initial event or subsequent tissue injury, which preferentiallyresults in tissue degradation.

Open angle glaucoma (OAG) the second led cause of irreversible blindnessin the United States, comprises 2 major syndromes: pi open angleglaucoma (PiOAG) and normal pressure glaucoma (NPG). PiOAG is a diseasegenerally characterized by a clinical triad which consists of 1)elevated IOP; 2) the appearance of optic atrophy presumably resultingfrom elevated IOP; and 3) a progressive loss of peripheral visualsensitivity in the early stages of the disease, which may ultimatelyprogress and impair central visual acuity (Quigley, 1993, New Engl J Med328:1097-1106.) Studies have indicated, however, that a surprisinglyhigh percentage of patients with OAG have findings identical to those inPiOAG, but with a singular exception; namely, that the IOP has neverbeen demonstrated to be elevated. Several large population-based studieshave documented the high prevalence of this form of glaucoma, oftencalled “low tension glaucoma” (but more accurately called NPG. The mostconservative of these estimates place the percentage of glaucoma thatoccurs in the presence of “normal” IOP at approximately 20-30% (Sommer,1989, Am J. Ophthalmol. 107:186-188; and Sommer, 1996, Eye 10:295-301).

In addition to the most common forms of glaucoma described above, thereare secondary and closed angle forms of glaucoma, which typically resultin elevated IOP due to a variety of mechanisms. In virtually all theseother forms of glaucoma, elevated eye pressure is found, and acharacteristic optic neuropathy similar to that found in OAG ensues. Ifuntreated, elevated intraocular pressure in these glaucomas invariablyleads to visual loss and eventual blindness. In many forms of glaucoma,including those with normal IOP, lowering of IOP often fails to halt theprogression of the disease. Comparison of glaucomatous progressionbetween untreated patients with normal-tension glaucoma and patientswith therapeutically reduced intraocular pressures (CollaborativeNormal-Tension Glaucoma Study Group, 1998, Am J. Ophthalmol.126:487-97).

Current anti-glaucoma therapies include lowering IOP by the use ofsuppressants of aqueous humor formation or agents that enhanceuveoscleral outflow, laser trabeculoplasty, or trabeculectomy, which isa filtration surgery to improve drainage. Pharmaceutical anti-glaucomaapproaches have exhibited various undesirable side effects. For example,miotics such as pilocarpine can cause blurring of vision and othernegative visual side effects. Systemically administered carbonicanhydrase inhibitors (CAIs) can also cause nausea, dyspepsia, fatigue,and metabolic acidosis. Further, certain beta-blockers have increasinglybecome associated with serious pulmonary side effects attributable totheir effects on beta-2 receptors in pulmonary tissue. Sympathomimeticscause tachycardia, arrhythmia and hypertension. Such negative sideeffects may lead to decreased patient compliance or to termination oftherapy. In addition, the efficacy of current IOP lowering therapies isrelatively short-lived requiring repeated dosing during each day and, insome cases, the efficacy decreases with time.

In view of the importance of glaucoma, and the inadequacies of priormethods of treatment, it would be desirable to have an improved methodof treatment.

Tumor necrosis factor α (TNFα) is a major mediator of the inflammatoryresponse, and has been implicated in many human diseases. Binding ofTNFα to its cell surface receptor, TNF receptor-1 (TNFR1), activates asignaling cascase affecting a wide variety of cellular responses,including apoptosis and inflammation. TNFα itself is initially expressedas an inactive, membrane-bound precursor. Release of the active form ofTNFα from the cell surface requires proteolytic processing of theprecursor by TNFα converting enzyme (TACE/ADAM17). Inhibiting expressionof TNFR1, TACE, or both will effectively reduce the action of TNFα.

In addition, studies have implicated TNFα signaling in the diseaseprocess in glaucoma. Retinal ganglion cells are susceptible toTNFα-induced apoptosis (Fuchs et al., 2005, Invest. Ophthalmol. Vis.Sci. 46:2983-2991). Expression of TNFα and its receptor, TNFR1, isincreased in glaucomatous eyes (Tezel et al., 2001, Invest. Ophthalmol.Vis. Sci. 42:1787-1794; Yuan et al., 2000, Glia 32:42-50; Yan et al.,2000, Arch. Ophthalmol. 118:666-673). In addition, U.S. Pat. No.6,531,128 showed increased expression of TNFα and TNFR1 in glaucomatousoptic nerve head and retina, suggesting a role for TNFα in theneurodegenerative process of glaucoma. In response to simulated ischemiaand increased hydrostatic pressure, glial cells secrete TNFα andfacilitate the apoptotic death of co-cultured retinal ganglion cells(Tezel et al., 2000, J. Neurosci. 20:8693-8700). Polymorphisms in theTNFα promoter are associated with increased risk for glaucoma (Lin etal., 2003, Eye 17:31-34; Funayama et al., 2004, Invest. Ophthalmol. Vis.Sci. 45:4359-4367). Thus, interfering with TNFα signaling is desirableto protect retinal ganglion cells from transient increases inintraocular pressure (IOP), and for treating and/or preventing elevatedIOP.

The present invention addresses the above-cited ocular pathologies andprovides compositions and methods using interfering RNAs that targetTACE and/or TNFR1 for treating ocular conditions associated withelevated IOP, such as glaucoma. U.S. Patent Publication 2005/0227935,published Oct. 13, 2005, to McSwiggen et al. relates to RNA interferencemediated inhibition of TNF and TNF receptor gene expression. However,said publication teaches none of the particular target sequences for RNAinterference as provided herein.

SUMMARY OF THE INVENTION

The invention provides interfering RNAs that silence expression of TACEmRNA or TNFR1 mRNA, thus interfering with proteolytic processing of theprecursor to TNFα, or interfering with binding of TNFα to its cellsurface receptor, respectively, thereby attenuating activity of TNFα,and decreasing TNFR1 or TACE levels in patients with a TNFα-relatedocular disorder or at risk of developing a TNFα-related ocular disorderassociated with elevated intraocular pressure (IOP). The interferingRNAs of the invention are useful for treating patients with elevatedIOP, such as ocular hypertension and glaucoma.

The invention also provides a method of attenuating expression of aTNFR1 or TACE mRNA in a subject. In one aspect, the method comprisesadministering to the subject a composition comprising an effectiveamount of interfering RNA having a length of 19 to 49 nucleotides and apharmaceutically acceptable carrier. In another aspect, administrationis to an eye of the subject for attenuating expression of TNFR1 or TACEin a human.

In one aspect, the invention provides a method of attenuating expressionof TACE mRNA in an eye of a subject, comprising administering to the eyeof the subject an interfering RNA that comprises a region that canrecognize a portion of mRNA corresponding to SEQ ID NO: 1, which is thesense cDNA sequence encoding TACE (GenBank Accession No. NM_(—)003183),wherein the expression of TACE mRNA is attenuated thereby. In addition,the invention provides methods of treating an TNFα-related oculardisorder in a subject in need thereof, comprising administering to theeye of the subject an interfering RNA that comprises a region that canrecognize a portion of mRNA corresponding to a portion of SEQ ID NO: 1,wherein the expression of TACE mRNA is attenuated thereby.

The invention also provides a method of attenuating expression of TNFR1mRNA in an eye of a subject, comprising administering to the eye of thesubject an interfering RNA that comprises a region that can recognize aportion of mRNA corresponding to SEQ ID NO: 2, which is the sense cDNAsequence encoding TNFR1 (GenBank Accession No. NM_(—)001065), whereinthe expression of TNFR1 mRNA is attenuated thereby. In addition, theinvention provides methods of treating an TNFα-related ocular disorderin a subject in need thereof, comprising administering to the eye of thesubject an interfering RNA that comprises a region that can recognize aportion of mRNA corresponding to a portion of SEQ ID NO: 2, wherein theexpression of TNFR1 mRNA is attenuated thereby.

In certain aspects, an interfering RNA of the invention is designed totarget an mRNA corresponding to a portion of SEQ ID NO: 1, wherein theportion comprises nucleotide 297, 333, 334, 335, 434, 470, 493, 547,570, 573, 618, 649, 689, 755, 842, 844, 846, 860, 878, 894, 900, 909,910, 913, 942, 970, 984, 1002, 1010, 1053, 1064, 1137, 1162, 1215, 1330,1334, 1340, 1386, 1393, 1428, 1505, 1508, 1541, 1553, 1557, 1591, 1592,1593, 1597, 1604, 1605, 1626, 1632, 1658, 1661, 1691, 1794, 1856, 1945,1946, 1947, 1958, 2022, 2094, 2100, 2121, 2263, 2277, 2347, 2349, 2549,2578, 2595, 2606, 2608, 2629, 2639, 2764, 2766, 2767, 2769, 3027, 3028,3261, 3264, 3284, 3313, 3317, 3332, or 3337 of SEQ ID NO: 1. Inparticular aspects, a “portion of SEQ ID NO: 1” is about 19 to about 49nucleotides in length.

In certain aspects, an interfering RNA of the invention is designed totarget an mRNA corresponding to a portion of SEQ ID NO: 2, wherein theportion comprises nucleotide 124, 328, 387, 391, 393, 395, 406, 421,423, 444, 447, 455, 459, 460, 467, 469, 470, 471, 475, 479, 513, 517,531, 543, 556, 576, 587, 588, 589, 595, 601, 602, 611, 612, 651, 664,667, 668, 669, 677, 678, 785, 786, 788, 791, 792, 804, 813, 824, 838,843, 877, 884, 929, 959, 960, 961, 963, 964, 965, 970, 973, 974, 1000,1002, 1013, 1026, 1053, 1056, 1057, 1058, 1161, 1315, 1318, 1324, 1357,1360, 1383, 1393, 1420, 1471, 1573, 1671, 2044, 2045, 2046, 2047, 2048,2089, 2090, 2091, or, 2092 of SEQ ID NO: 2. In particular aspects, a“portion of SEQ ID NO: 2” is about 19 to about 49 nucleotides in length.

In certain aspects, an interfering RNA of the invention has a length ofabout 19 to about 49 nucleotides. In other aspects, the interfering RNAcomprises a sense nucleotide strand and an antisense nucleotide strand,wherein each strand has a region of at least near-perfect contiguouscomplementarity of at least 19 nucleotides with the other strand, andwherein the antisense strand can recognize (a) a portion of TACE mRNAcorresponding to a portion of SEQ ID NO: 1, and has a region of at leastnear-perfect contiguous complementarity of at least 19 nucleotides withthe portion of TACE mRNA; or (b) a portion of TNFR1 mRNA correspondingto a portion of SEQ ID NO: 2, and has a region of at least near-perfectcontiguous complementarity of at least 19 nucleotides with the portionof TNFR1 mRNA. The sense and antisense strands can be connected by alinker sequence, which allows the sense and antisense strands tohybridize to each other thereby forming a hairpin loop structure asdescribed herein.

In still other aspects, an interfering RNA of the invention is asingle-stranded interfering RNA, and wherein single-stranded interferingRNA recognizes a portion of mRNA corresponding to a portion of SEQ IDNO: 1 or SEQ ID NO: 2. In certain aspects, the interfering RNA has aregion of at least near-perfect contiguous complementarity of at least19 nucleotides with the portion of mRNA corresponding to the portion ofSEQ ID NO: 1 or SEQ ID NO: 2. In other aspects, the portion of SEQ IDNO: 1 comprises 297, 333, 334, 335, 434, 470, 493, 547, 570, 573, 618,649, 689, 755, 842, 844, 846, 860, 878, 894, 900, 909, 910, 913, 942,970, 984, 1002, 1010, 1053, 1064, 1137, 1162, 1215, 1330, 1334, 1340,1386, 1393, 1428, 1505, 1508, 1541, 1553, 1557, 1591, 1592, 1593, 1597,1604, 1605, 1626, 1632, 1658, 1661, 1691, 1794, 1856, 1945, 1946, 1947,1958, 2022, 2094, 2100, 2121, 2263, 2277, 2347, 2349, 2549, 2578, 2595,2606, 2608, 2629, 2639, 2764, 2766, 2767, 2769, 3027, 3028, 3261, 3264,3284, 3313, 3317, 3332, or 3337 of SEQ ID NO: 1. In other aspects, theportion of SEQ ID NO: 2 comprises 124, 328, 387, 391, 393, 395, 406,421, 423, 444, 447, 455, 459, 460, 467, 469, 470, 471, 475, 479, 513,517, 531, 543, 556, 576, 587, 588, 589, 595, 601, 602, 611, 612, 651,664, 667, 668, 669, 677, 678, 785, 786, 788, 791, 792, 804, 813, 824,838, 843, 877, 884, 929, 959, 960, 961, 963, 964, 965, 970, 973, 974,1000, 1002, 1013, 1026, 1053, 1056, 1057, 1058, 1161, 1315, 1318, 1324,1357, 1360, 1383, 1393, 1420, 1471, 1573, 1671, 2044, 2045, 2046, 2047,2048, 2089, 2090, 2091, or, 2092 of SEQ ID NO: 2.

In still other aspects, an interfering RNA of the invention comprises:(a) a region of at least 13 contiguous nucleotides having at least 90%sequence complementarity to, or at least 90% sequence identity with, thepenultimate 13 nucleotides of the 3′ end of a mRNA corresponding to anyone of SEQ ID NO:3 and SEQ ID NO:14-SEQ ID NO:58; (b) a region of atleast 14 contiguous nucleotides having at least 85% sequencecomplementarity to, or at least 85% sequence identity with, thepenultimate 14 nucleotides of the 3′ end of an mRNA corresponding to anyone of SEQ ID NO:3 and SEQ ID NO:14-SEQ ID NO:58; or (c) a region of atleast 15, 16, 17, or 18 contiguous nucleotides having at least 80%sequence complementarity to, or at least 80% sequence identity with, thepenultimate 15, 16, 17, or 18 nucleotides, respectively, of the 3′ endof an mRNA corresponding to any one of SEQ ID NO:3 and SEQ ID NO:14-SEQID NO:58; wherein the expression of the TACE mRNA is attenuated thereby.

In still other aspects, an interfering RNA of the invention comprises:(a) a region of at least 13 contiguous nucleotides having at least 90%sequence complementarity to, or at least 90% sequence identity with, thepenultimate 13 nucleotides of the 3′ end of a mRNA corresponding to anyone of SEQ ID NO:155-SEQ ID NO:201; (b) a region of at least 14contiguous nucleotides having at least 85% sequence complementarity to,or at least 85% sequence identity with, the penultimate 14 nucleotidesof the 3′ end of an mRNA corresponding to any one of SEQ ID NO:155-SEQID NO:201; or (c) a region of at least 15, 16, 17, or 18 contiguousnucleotides having at least 80% sequence complementarity to, or at least80% sequence identity with, the penultimate 15, 16, 17, or 18nucleotides, respectively, of the 3′ end of an mRNA corresponding to anyone of SEQ ID NO:155-SEQ ID NO:201; wherein the expression of the TNFR1mRNA is attenuated thereby.

In further aspects, an interfering RNA of the invention or compositioncomprising an interfering RNA of the invention is administered to asubject via a topical, intravitreal, transcleral, periocular,conjunctival, subtenon, intracameral, subretinal, subconjunctival,retrobulbar, or intracanalicular route. The interfering RNA orcomposition can be administered, for example, via in vivo expressionfrom an interfering RNA expression vector. In certain aspects, theinterfering RNA or composition can be administered via an aerosol,buccal, dermal, intradermal, inhaling, intramuscular, intranasal,intraocular, intrapulmonary, intravenous, intraperitoneal, nasal,ocular, oral, otic, parenteral, patch, subcutaneous, sublingual,topical, or transdermal route.

In one aspect, an interfering RNA molecule of the invention is isolated.The term “isolated” means that the interfering RNA is free of its totalnatural milieu.

The invention further provides methods of treating a TNFα-related oculardisorder in a subject in need thereof, comprising administering to thesubject a composition comprising a double-stranded siRNA molecule thatdown regulates expression of a TACE or TNFR1 gene via RNA interference,wherein each strand of the siRNA molecule is independently about 19 toabout 27 nucleotides in length, and one strand of the siRNA moleculecomprises a nucleotide sequence having substantial complementarity to anmRNA corresponding to the TACE or TNFR1 gene so that the siRNA moleculedirects cleavage of the mRNA via RNA interference. In certain aspects,the siRNA molecule is administered via an aerosol, buccal, dermal,intradermal, inhaling, intramuscular, intranasal, intraocular,intrapulmonary, intravenous, intraperitoneal, nasal, ocular, oral, otic,parenteral, patch, subcutaneous, sublingual, topical, or transdermalroute.

The invention further provides for administering a second interferingRNA to a subject in addition to a first interfering RNA. The secondinterfering RNA may target the same mRNA target gene as the firstinterfering RNA or may target a different gene. Further, a third,fourth, or fifth, etc. interfering RNA may be administered in a similarmanner.

Use of any of the embodiments as described herein in the preparation ofa medicament for attenuating expression of TACE or TNFR1 mRNA is also anembodiment of the present invention.

Specific preferred embodiments of the invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a TNFR1 western blot of GTM-3 cells transfected withTNFR1 siRNAs #1, #2, #3, and #4, and a RISC-free control siRNA, each at10 nM, 1 nM, and 0.1 nM; a non-targeting control siRNA (NTC2) at 10 nM;and a buffer control (-siRNA). The arrows indicate the positions of the55-kDa TNFR1 and 42-kDa actin bands.

DETAILED DESCRIPTION OF THE INVENTION

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

The following definitions and explanations are meant and intended to becontrolling in any future construction unless clearly and unambiguouslymodified in the following examples or when application of the meaningrenders any construction meaningless or essentially meaningless. Incases where the construction of the term would render it meaningless oressentially meaningless, the definition should be taken from Webster'sDictionary, 3^(rd) Edition or a dictionary known to those of skill inthe art, such as the Oxford Dictionary of Biochemistry and MolecularBiology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

As used herein, all percentages are percentages by weight, unless statedotherwise.

As used herein and unless otherwise indicated, the terms “a” and “an”are taken to mean “one”, “at least one” or “one or more”. Unlessotherwise required by context, singular terms used herein shall includepluralities and plural terms shall include the singular.

In certain embodiments, the invention provides interfering RNA moleculesthat can direct cleavage and/or degradation of TNFα cell surfacereceptor TNF receptor-1 (TNFR1) mRNA, or the TNFα converting enzyme(TACE/ADAM17, designated herein “TACE”) mRNA, which inhibition effectsreduction of tumor necrosis factor α (TNFα) activity, via RNAinterference. Binding of TNFα to its cell surface receptor, TNFreceptor-1 (TNFR1), activates a signaling cascade which affects avariety of cellular responses including apoptosis and inflammation. TNFαitself is initially expressed as an inactive, membrane-bound precursor.Release of the active form of TNFα from the cell surface requiresproteolytic processing of the precursor by TNFα converting enzyme(TACE/ADAM17), a member of the ‘A Disintegrin And Metalloprotease’(ADAM) family.

According to the present invention, inhibiting the expression of TNFR1mRNA, TACE mRNA, or both TNFR1 and TACE mRNAs effectively reduces theaction of TNFα. Further, interfering RNAs as set forth herein providedexogenously or expressed endogenously are particularly effective atsilencing TNFR1 mRNA or TACE mRNA.

RNA interference (RNAi) is a process by which double-stranded RNA(dsRNA) is used to silence gene expression. While not wanting to bebound by theory, RNAi begins with the cleavage of longer dsRNAs intosmall interfering RNAs (siRNAs) by an RNaseIII-like enzyme, dicer.SiRNAs are dsRNAs that are usually about 19 to 28 nucleotides, or 20 to25 nucleotides, or 21 to 22 nucleotides in length and often contain2-nucleotide 3′ overhangs, and 5′ phosphate and 3′ hydroxyl termini. Onestrand of the siRNA is incorporated into a ribonucleoprotein complexknown as the RNA-induced silencing complex (RISC). RISC uses this siRNAstrand to identify mRNA molecules that are at least partiallycomplementary to the incorporated siRNA strand, and then cleaves thesetarget mRNAs or inhibits their translation. Therefore, the siRNA strandthat is incorporated into RISC is known as the guide strand or theantisense strand. The other siRNA strand, known as the passenger strandor the sense strand, is eliminated from the siRNA and is at leastpartially homologous to the target mRNA. Those of skill in the art willrecognize that, in principle, either strand of an siRNA can beincorporated into RISC and function as a guide strand. However, siRNAdesign (e.g., decreased siRNA duplex stability at the 5′ end of thedesired guide strand) can favor incorporation of the desired guidestrand into RISC.

The antisense strand of an siRNA is the active guiding agent of thesiRNA in that the antisense strand is incorporated into RISC, thusallowing RISC to identify target mRNAs with at least partialcomplementarity to the antisense siRNA strand for cleavage ortranslational repression. RISC-related cleavage of mRNAs having asequence at least partially complementary to the guide strand leads to adecrease in the steady state level of that mRNA and of the correspondingprotein encoded by this mRNA. Alternatively, RISC can also decreaseexpression of the corresponding protein via translational repressionwithout cleavage of the target mRNA.

Interfering RNAs of the invention appear to act in a catalytic mannerfor cleavage of target mRNA, i.e., interfering RNA is able to effectinhibition of target mRNA in substoichiometric amounts. As compared toantisense therapies, significantly less interfering RNA is required toprovide a therapeutic effect under such cleavage conditions.

In certain embodiments, the invention provides methods of usinginterfering RNA to inhibit the expression of TACE or TNFR1 target mRNAthus decreasing TACE or TNFR1 levels in patients with a TNFα-relatedocular disorder. According to the present invention, interfering RNAsprovided exogenously or expressed endogenously effect silencing of TACEor TNFR1 expression in ocular tissues.

The phrase, “attenuating expression of an mRNA,” as used herein, meansadministering or expressing an amount of interfering RNA (e.g., ansiRNA) to reduce translation of the target mRNA into protein, eitherthrough mRNA cleavage or through direct inhibition of translation. Theterms “inhibit,” “silencing,” and “attenuating” as used herein refer toa measurable reduction in expression of a target mRNA or thecorresponding protein as compared with the expression of the target mRNAor the corresponding protein in the absence of an interfering RNA of theinvention. The reduction in expression of the target mRNA or thecorresponding protein is commonly referred to as “knock-down” and isreported relative to levels present following administration orexpression of a non-targeting control RNA (e.g., a non-targeting controlsiRNA). Knock-down of expression of an amount including and between 50%and 100% is contemplated by embodiments herein. However, it is notnecessary that such knock-down levels be achieved for purposes of thepresent invention.

Knock-down is commonly assessed by measuring the mRNA levels usingquantitative polymerase chain reaction (qPCR) amplification or bymeasuring protein levels by western blot or enzyme-linked immunosorbentassay (ELISA). Analyzing the protein level provides an assessment ofboth mRNA cleavage as well as translation inhibition. Further techniquesfor measuring knock-down include RNA solution hybridization, nucleaseprotection, northern hybridization, gene expression monitoring with amicroarray, antibody binding, radioimmunoassay, and fluorescenceactivated cell analysis.

Attenuating expression of TACE or TNFR1 by an interfering RNA moleculeof the invention can be inferred in a human or other mammal by observingan improvement in a glaucoma symptom such as improvement in intraocularpressure, improvement in visual field loss, or improvement in opticnerve head changes, for example.

The ability of TACE- or TNFR1-interfering RNA to knock-down the levelsof TACE or TNFR1 gene expression in, for example, HeLa cells can beevaluated in vitro as follows. HeLa cells are plated 24 h prior totransfection in standard growth medium (e.g., DMEM supplemented with 10%fetal bovine serum). Transfection is performed using, for example,Dharmafect 1 (Dharmacon, Lafayette, Colo.) according to themanufacturer's instructions at interfering RNA concentrations rangingfrom 0.1 nM-100 nM. SiCONTROL™ Non-Targeting siRNA #1 and siCONTROL™Cyclophilin B siRNA (Dharmacon) are used as negative and positivecontrols, respectively. Target mRNA levels and cyclophilin B mRNA (PPIB,NM_(—)000942) levels are assessed by qPCR 24 h post-transfection using,for example, a TAQMAN® Gene Expression Assay that preferably overlapsthe target site (Applied Biosystems, Foster City, Calif.). The positivecontrol siRNA gives essentially complete knockdown of cyclophilin B mRNAwhen transfection efficiency is 100%. Therefore, target mRNA knockdownis corrected for transfection efficiency by reference to the cyclophilinB mRNA level in cells transfected with the cyclophilin B siRNA. Targetprotein levels may be assessed approximately 72 h post-transfection(actual time dependent on protein turnover rate) by western blot, forexample. Standard techniques for RNA and/or protein isolation fromcultured cells are well-known to those skilled in the art. To reduce thechance of non-specific, off-target effects, the lowest possibleconcentration of interfering RNA is used that produces the desired levelof knock-down in target gene expression.

Human trabecular meshwork (TM) cells or other human ocular cell linesmay also be use for an evaluation of the ability of interfering RNA toknock-down levels of an endogenous target gene. The ability of TACE- orTNFR1-interfering RNA to knock-down the levels of endogenous TACE orTNFR1 expression in, for example, human GTM-3 cells (Pang et al., 1994,Curr. Eye Res. 13:51-63) can be evaluated in vitro as follows. GTM-3cells are plated 24 h prior to transfection in DMEM medium supplementedwith 10% FBS and 2 mM glutamate. Transfection is performed usingDharmaFECT™ 1 (Dharmacon, Lafayette, Colo.) according to themanufacturer's instructions at TACE- or TNFR1-interfering RNAconcentrations ranging from 0.1 nM-100 nM. Non-targeting controlinterfering RNA and cyclophilin B interfering RNA are used as controls.Target mRNA levels are assessed by qPCR 24 h post-transfection using,for example, TAQMAN® forward and reverse primers and a probe set thatencompasses the target site (Applied Biosystems, Foster City, Calif.).Target protein levels may be assessed approximately 72 hpost-transfection (actual time dependent on protein turnover rate) bywestern blot, for example. Standard techniques for RNA and/or proteinisolation from cultured cells are well-known to those skilled in theart. To reduce the chance of non-specific, off-target effects, thelowest possible concentration of TACE- or TNFR1 interfering RNA is usedthat produces the desired level of knock-down in target gene expression.

In one embodiment, a single interfering RNA targeting TACE or TNFR1 mRNAis administered to decrease TACE or TNFR1 levels. In other embodiments,two or more interfering RNAs targeting the TACE and/or TNFR1 mRNA areadministered to decrease TACE and/or TNFR1 levels. In certainembodiments, interfering RNA targeting TACE and interfering RNAtargeting TNFR1 are administered to the subject sequentially orconcurrently, thereby treating the TNFα-related ocular disease.

The GenBank database provides the DNA sequence for TACE as accession no.NM_(—)003183, provided in the “Sequence Listing” as SEQ ID NO:1. SEQ IDNO:1 provides the sense strand sequence of DNA that corresponds to themRNA encoding TACE (with the exception of “T” bases for “U” bases). Thecoding sequence for TACE is from nucleotides 184-2658.

Equivalents of the above cited TACE mRNA sequence are alternative spliceforms, allelic forms, isozymes, or a cognate thereof. A cognate is atumor necrosis factor α converting enzyme mRNA from another mammalianspecies that is homologous to SEQ ID NO:1 (i.e., an ortholog).

The GenBank database provides the DNA sequence for TNFR1 as accessionno. NM_(—)001065, provided in the “Sequence Listing” as SEQ ID NO:2. SEQID NO:2 provides the sense strand sequence of DNA that corresponds tothe mRNA encoding TNFR1 (with the exception of “T” bases for “U” bases).The coding sequence for TNFR1 is from nucleotides 282-1649.

Equivalents of the above cited TNFR1 mRNA sequence are alternativesplice forms, allelic forms, isozymes, or a cognate thereof. A cognateis a tumor necrosis factor receptor-1 mRNA from another mammalianspecies that is homologous to SEQ ID NO:2 (i.e., an ortholog).

In certain embodiments, a “subject” in need of treatment for aTNFα-related ocular disorder or at risk for developing a TNFα-relatedocular disorder is a human or other mammal having a TNFα-related oculardisorder or at risk of having a TNFα-related ocular disorder associatedwith undesired or inappropriate expression or activity of targets ascited herein, i.e., TACE or TNFR1. Ocular structures associated withsuch disorders may include the eye, retina, choroid, lens, cornea,trabecular meshwork, iris, optic nerve, optic nerve head, sclera,anterior or posterior segment, or ciliary body, for example. A subjectmay also be an ocular cell, cell culture, organ or an ex vivo organ ortissue or cell. In certain embodiments, a subject has or is at risk fordeveloping ocular hypertension and/or ocular diseases associated withelevated intraocular pressure (IOP), such as glaucoma.

“TNFα-related ocular disorder” as used herein includes ocularhypertension and ocular diseases associated with elevated intraocularpressure (IOP), such as glaucoma.

The term “siRNA” as used herein refers to a double-stranded interferingRNA unless otherwise noted. Typically, an siRNA of the invention is adouble-stranded nucleic acid molecule comprising two nucleotide strands,each strand having about 19 to about 28 nucleotides (i.e. about 19, 20,21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). The phrase “interferingRNA having a length of 19 to 49 nucleotides” when referring to adouble-stranded interfering RNA means that the antisense and sensestrands independently have a length of about 19 to about 49 nucleotides,including interfering RNA molecules where the sense and antisensestrands are connected by a linker molecule.

In addition to siRNA molecules, other interfering RNA molecules andRNA-like molecules can interact with RISC and silence gene expression.Examples of other interfering RNA molecules that can interact with RISCinclude short hairpin RNAs (shRNAs), single-stranded siRNAs, microRNAs(miRNAs), and dicer-substrate 27-mer duplexes. Examples of RNA-likemolecules that can interact with RISC include siRNA, single-strandedsiRNA, microRNA, and shRNA molecules containing one or more chemicallymodified nucleotides, one or more non-nucleotides, one or moredeoxyribonucleotides, and/or one or more non-phosphodiester linkages.All RNA or RNA-like molecules that can interact with RISC andparticipate in RISC-related changes in gene expression are referred toherein as “interfering RNAs” or “interfering RNA molecules.” SiRNAs,single-stranded siRNAs, shRNAs, miRNAs, and dicer-substrate 27-merduplexes are, therefore, subsets of “interfering RNAs” or “interferingRNA molecules.”

Single-stranded interfering RNA has been found to effect mRNA silencing,albeit less efficiently than double-stranded RNA. Therefore, embodimentsof the present invention also provide for administration of asingle-stranded interfering RNA that has a region of at leastnear-perfect contiguous complementarity with a portion of SEQ ID NO: 1or a portion of SEQ ID NO: 2. The single-stranded interfering RNA has alength of about 19 to about 49 nucleotides as for the double-strandedinterfering RNA cited above. The single-stranded interfering RNA has a5′ phosphate or is phosphorylated in situ or in vivo at the 5′ position.The term “5′ phosphorylated” is used to describe, for example,polynucleotides or oligonucleotides having a phosphate group attachedvia ester linkage to the C5 hydroxyl of the sugar (e.g., ribose,deoxyribose, or an analog of same) at the 5′ end of the polynucleotideor oligonucleotide.

Single-stranded interfering RNAs can be synthesized chemically or by invitro transcription or expressed endogenously from vectors or expressioncassettes as described herein in reference to double-strandedinterfering RNAs. 5′ Phosphate groups may be added via a kinase, or a 5′phosphate may be the result of nuclease cleavage of an RNA. A hairpininterfering RNA is a single molecule (e.g., a single oligonucleotidechain) that comprises both the sense and antisense strands of aninterfering RNA in a stem-loop or hairpin structure (e.g., a shRNA). Forexample, shRNAs can be expressed from DNA vectors in which the DNAoligonucleotides encoding a sense interfering RNA strand are linked tothe DNA oligonucleotides encoding the reverse complementary antisenseinterfering RNA strand by a short spacer. If needed for the chosenexpression vector, 3′ terminal T's and nucleotides forming restrictionsites may be added. The resulting RNA transcript folds back onto itselfto form a stem-loop structure.

Nucleic acid sequences cited herein are written in a 5′ to 3′ directionunless indicated otherwise. The term “nucleic acid,” as used herein,refers to either DNA or RNA or a modified form thereof comprising thepurine or pyrimidine bases present in DNA (adenine “A,” cytosine “C,”guanine “G,” thymine “T”) or in RNA (adenine “A,” cytosine “C,” guanine“G,” uracil “U”). Interfering RNAs provided herein may comprise “T”bases, particularly at 3′ ends, even though “T” bases do not naturallyoccur in RNA. “Nucleic acid” includes the terms “oligonucleotide” and“polynucleotide” and can refer to a single-stranded molecule or adouble-stranded molecule. A double-stranded molecule is formed byWatson-Crick base pairing between A and T bases, C and G bases, andbetween A and U bases. The strands of a double-stranded molecule mayhave partial, substantial or full complementarity to each other and willform a duplex hybrid, the strength of bonding of which is dependent uponthe nature and degree of complementarity of the sequence of bases.

The phrase “DNA target sequence” as used herein refers to the DNAsequence that is used to derive an interfering RNA of the invention. Thephrases “RNA target sequence,” “interfering RNA target sequence,” and“RNA target” as used herein refer to the TACE or TNFR1 mRNA or theportion of the TACE or TNFR1 mRNA sequence that can be recognized by aninterfering RNA of the invention, whereby the interfering RNA cansilence TACE or TNFR1 gene expression as discussed herein. An “RNAtarget sequence,” an “siRNA target sequence,” and an “RNA target” aretypically mRNA sequences that correspond to a portion of a DNA sequence.An mRNA sequence is readily deduced from the sequence of thecorresponding DNA sequence. For example, SEQ ID NO: 1 provides the sensestrand sequence of DNA corresponding to the mRNA for TACE, while SEQ IDNO: 2 provides the sense strand sequence of DNA corresponding to themRNA for TNFR1. The mRNA sequence is identical to the DNA sense strandsequence with the “T” bases replaced with “U” bases. Therefore, the mRNAsequence of TACE is known from SEQ ID NO: 1, and the mRNA sequence ofTNFR1 is known from SEQ ID NO: 2. A target sequence in the mRNAscorresponding to SEQ ID NO: 1 or SEQ ID NO: 2 may be in the 5′ or 3′untranslated regions of the mRNA as well as in the coding region of themRNA.

In certain embodiments, interfering RNA target sequences (e.g., siRNAtarget sequences) within a target mRNA sequence are selected usingavailable design tools. Interfering RNAs corresponding to a TACE orTNFR1 target sequence are then tested in vitro by transfection of cellsexpressing the target mRNA followed by assessment of knockdown asdescribed herein. The interfering RNAs can be further evaluated in vivousing animal models as described herein.

Techniques for selecting target sequences for siRNAs are provided, forexample, by Tuschl, T. et al., “The siRNA User Guide,” revised May 6,2004, available on the Rockefeller University web site; by TechnicalBulletin #506, “siRNA Design Guidelines,” Ambion Inc. at Ambion's website; and by other web-based design tools at, for example, theInvitrogen, Dharmacon, Integrated DNA Technologies, Genscript, orProligo web sites. Initial search parameters can include G/C contentsbetween 35% and 55% and siRNA lengths between 19 and 27 nucleotides. Thetarget sequence may be located in the coding region or in the 5′ or 3′untranslated regions of the mRNA. The target sequences can be used toderive interfering RNA molecules, such as those described herein.

Table 1 lists examples of TACE DNA target sequences of SEQ ID NO: 1 fromwhich interfering RNA molecules of the present invention are designed ina manner as set forth above.

TABLE 1 TACE Target Sequences for siRNAs # of Starting Nucleotide withreference to TACE Target Sequence SEQ ID NO: 1 SEQ ID NO:GCTCTCAGACTACGATATT 297 3 CCAGCAGCATTCGGTAAGA 333 14 CAGCAGCATTCGGTAAGAA334 15 AGCAGCATTCGGTAAGAAA 335 16 AGAGATCTACAGACTTCAA 355 17GAAAGCGAGTACACTGTAA 493 18 CCATGAAGAACACGTGTAA 842 19GAAGAACACGTGTAAATTA 846 20 ATCATCGCTTCTACAGATA 878 21AGAGCAATTTAGCTTTGAT 1137 22 GGTTTGACGAGCACAAAGA 1330 23TGATCCGGATGGTCTAGCA 1428 24 GCGATCACGAGAACAATAA 1508 25GCAGTAAACAATCAATCTA 1541 26 CAATCTATAAGACCATTGA 1553 27TTTCAAGAACGCAGCAATA 1591 28 TTCAAGAACGCAGCAATAA 1592 29TCAAGAACGCAGCAATAAA 1593 30 TCATGTATCTGAACAACGA 1661 31ACAGCGACTGCACGTTGAA 1691 32 GATTAATGCTACTTGCAAA 1794 33CTGGAGTCCTGTGCATGTA 1945 34 TGGAGTCCTGTGCATGTAA 1946 35GGAGTCCTGTGCATGTAAT 1947 36 CATGTAATGAAACTGACAA 1958 37CTATGTCGATGCTGAACAA 2022 38 CAAATGTGAGAAACGAGTA 2100 39GCATCGGTTCGCATTATCA 2347 40 ATCGGTTCGCATTATCAAA 2349 41CCAAGTCATTTGAGGATCT 2549 42 CCGGTCACCAGAAGTGAAA 2578 43AAAGGCTGCCTCCTTTAAA 2595 44 TTTAAACTGCAGCGTCAGA 2608 45AGATGCTGGTCATGTGTTT 2764 46 ATGCTGGTCATGTGTTTGA 2766 47TGCTGGTCATGTGTTTGAA 2767 48 CTGGTCATGTGTTTGAACT 2769 49TGTAATGAACCGCTGAATA 3027 50 GTAATGAACCGCTGAATAT 3028 51CTAAGACTAATGCTCTCTA 3261 52 AGACTAATGCTCTCTAGAA 3264 53CCTAACCACCTACCTTACA 3284 54 TACATGGTAGCCAGTTGAA 3313 55TGGTAGCCAGTTGAATTTA 3317 56 TTTATGGAATCTACCAACT 3332 57GGAATCTACCAACTGTTTA 3337 58 CATCAAGTACTGAACGTTT 434 155TCGTGGTGGTGGATGGTAA 470 156 GAAAGCGAGTACACTGTAA 493 157GAGCCTGACTCTAGGGTTC 547 158 CCACATAAGAGATGATGAT 570 159CATAAGAGATGATGATGTT 573 160 CGAATATAACATAGAGCCA 618 161GTTAATGATACCAAAGACA 649 162 CTGAAGATATCAAGAATGT 689 163ATGAAGAGTTGCTCCCAAA 755 164 ATGAAGAACACGTGTAAAT 844 165AATTATTGGTGGTAGCAGA 860 166 ATCATCGCTTCTACAGATA 878 167ATACATGGGCAGAGGGGAA 894 168 GGGCAGAGGGGAAGAGAGT 900 169GGAAGAGAGTACAACTACA 909 170 GAAGAGAGTACAACTACAA 910 171GAGAGTACAACTACAAATT 913 172 GCTAATTGACAGAGTTGAT 942 173CGGAACACTTCATGGGATA 970 174 GGATAATGCAGGTTTTAAA 984 175AGGCTATGGAATACAGATA 1002 176 GAATACAGATAGAGCAGAT 1010 177GGTAAAACCTGGTGAAAAG 1053 178 GTGAAAAGCACTACAACAT 1064 179GAGGAAGCATCTAAAGTTT 1162 180 TATGGGAACTCTTGGATTA 1215 181TGACGAGCACAAAGAATTA 1334 182 GCACAAAGAATTATGGTAA 1340 183GGTTACAACTCATGAATTG 1386 184 ACTCATGAATTGGGACATA 1393 185GTGGCGATCACGAGAACAA 1505 186 CTATAAGACCATTGAAAGT 1557 187GAACGCAGCAATAAAGTTT 1597 188 GCAATAAAGTTTGTGGGAA 1604 189CAATAAAGTTTGTGGGAAC 1605 190 GAGGGTGGATGAAGGAGAA 1626 191GGATGAAGGAGAAGAGTGT 1632 192 GCATCATGTATCTGAACAA 1658 193CAGGAAATGCTGAAGATGA 1856 194 GAATGGCAAATGTGAGAAA 2094 195GGATGTAATTGAACGATTT 2121 196 GTGGATAAGAAATTGGATA 2263 197GGATAAACAGTATGAATCT 2277 198 CCTTTAAACTGCAGCGTCA 2606 199CGTGTTGACAGCAAAGAAA 2629 200 GCAAAGAAACAGAGTGCTA 2639 201

Table 2 lists examples of TNFR1 DNA target sequences of SEQ ID NO:2 fromwhich siRNAs of the present invention are designed in a manner as setforth above. TNFR1 encodes tumor necrosis factor α receptor-1, as notedabove.

TABLE 2 TNFR1 Target Sequences for siRNAs # of Starting Nucleotide withreference to TNFR1 Target Sequence SEQ ID NO: 2 SEQ ID NO:ACCAGGCCGTGATCTCTAT 124 59 AATTCGATTTGCTGTACCA 444 60TCGATTTGCTGTACCAAGT 447 61 ACAAAGGAACCTACTTGTA 469 62GAACCTACTTGTACAATGA 475 63 CTACTTGTACAATGACTGT 479 64TGTGAGAGCGGCTCCTTCA 531 65 TCAGGTGGAGATCTCTTCT 611 66CAGGTGGAGATCTCTTCTT 612 67 AGAACCAGTACCGGCATTA 667 68GAACCAGTACCGGCATTAT 668 69 AACCAGTACCGGCATTATT 669 70CCGGCATTATTGGAGTGAA 677 71 CGGCATTATTGGAGTGAAA 678 72AGCCTGGAGTGCACGAAGT 843 73 CTCCTCTTCATTGGTTTAA 960 74TTGGTTTAATGTATCGCTA 970 75 GTTTAATGTATCGCTACCA 973 76TTTAATGTATCGCTACCAA 974 77 AGTCCAAGCTCTACTCCAT 1000 78GAGCTTGAAGGAACTACTA 1053 79 CTTGAAGGAACTACTACTA 1056 80TTGAAGGAACTACTACTAA 1057 81 ACAAGCCACAGAGCCTAGA 1318 82TGTACGCCGTGGTGGAGAA 1357 83 CCGTTGCGCTGGAAGGAAT 1383 84TCTAAGGACCGTCCTGCGA 1671 85 CTAATAGAAACTTGGCACT 2044 86TAATAGAAACTTGGCACTC 2045 87 AATAGAAACTTGGCACTCC 2046 88ATAGAAACTTGGCACTCCT 2047 89 TAGAAACTTGGCACTCCTG 2048 90ATAGCAAGCTGAACTGTCC 2089 91 TAGCAAGCTGAACTGTCCT 2090 92AGCAAGCTGAACTGTCCTA 2091 93 GCAAGCTGAACTGTCCTAA 2092 94TGAACTGTCCTAAGGCAGG 2098 95 CAAAGGAACCTACTTGTAC 470 96GAGCTTGAAGGAACTACTA 1053 97 CACAGAGCCTAGACACTGA 1324 98TCCAAGCTCTACTCCATTG 1002 99 TGGAGCTGTTGGTGGGAAT 328 100GACAGGGAGAAGAGAGATA 387 101 GGGAGAAGAGAGATAGTGT 391 102GAGAAGAGAGATAGTGTGT 393 103 GAAGAGAGATAGTGTGTGT 395 104GTGTGTGTCCCCAAGGAAA 406 105 GAAAATATATCCACCCTCA 421 106AAATATATCCACCCTCAAA 423 107 CTGTACCAAGTGCCACAAA 455 108ACCAAGTGCCACAAAGGAA 459 109 CCAAGTGCCACAAAGGAAC 460 110CCACAAAGGAACCTACTTG 467 111 CAAAGGAACCTACTTGTAC 470 112AAAGGAACCTACTTGTACA 471 113 GATACGGACTGCAGGGAGT 513 114CGGACTGCAGGGAGTGTGA 517 115 TCCTTCACCGCTTCAGAAA 543 116CAGAAAACCACCTCAGACA 556 117 TGCCTCAGCTGCTCCAAAT 576 118CTCCAAATGCCGAAAGGAA 587 119 TCCAAATGCCGAAAGGAAA 588 120CCAAATGCCGAAAGGAAAT 589 121 GCCGAAAGGAAATGGGTCA 595 122AGGAAATGGGTCAGGTGGA 601 123 GGAAATGGGTCAGGTGGAG 602 124GTGTGTGGCTGCAGGAAGA 651 125 GGAAGAACCAGTACCGGCA 664 126CCATGCAGGTTTCTTTCTA 785 127 CATGCAGGTTTCTTTCTAA 786 128TGCAGGTTTCTTTCTAAGA 788 129 AGGTTTCTTTCTAAGAGAA 791 130GGTTTCTTTCTAAGAGAAA 792 131 AGAGAAAACGAGTGTGTCT 804 132GAGTGTGTCTCCTGTAGTA 813 133 CTGTAGTAACTGTAAGAAA 824 134AGAAAAGCCTGGAGTGCAC 838 135 TTGAGAATGTTAAGGGCAC 877 136TGTTAAGGGCACTGAGGAC 884 137 GGTCATTTTCTTTGGTCTT 929 138CCTCCTCTTCATTGGTTTA 959 139 TCCTCTTCATTGGTTTAAT 961 140CTCTTCATTGGTTTAATGT 963 141 TCTTCATTGGTTTAATGTA 964 142CTTCATTGGTTTAATGTAT 965 143 TCCAAGCTCTACTCCATTG 1002 144CTCCATTGTTTGTGGGAAA 1013 145 GGGAAATCGACACCTGAAA 1026 146TGAAGGAACTACTACTAAG 1058 147 ACCTCCAGCTCCACCTATA 1161 148CCCACAAGCCACAGAGCCT 1315 149 ACGCCGTGGTGGAGAACGT 1360 150GGAAGGAATTCGTGCGGCG 1393 151 TGAGCGACCACGAGATCGA 1420 152GCGAGGCGCAATACAGCAT 1471 153 TGGGCTGCCTGGAGGACAT 1573 154

As cited in the examples above, one of skill in the art is able to usethe target sequence information provided in Table 1 to designinterfering RNAs having a length shorter or longer than the sequencesprovided in Table 1 by referring to the sequence position in SEQ ID NO:1 and adding or deleting nucleotides complementary or near complementaryto SEQ ID NO: 1.

For example, SEQ ID NO: 3 represents a 19-nucleotide DNA target sequencefor TACE mRNA is present at nucleotides 297 to 315 of SEQ ID NO:1:

5′-GCTCTCAGACTACGATATT-3′. SEQ ID NO: 3

An example of an siRNA of the invention for targeting a correspondingmRNA sequence of SEQ ID NO:3 and having 21-nucleotide strands and a2-nucleotide 3′ overhang is:

5′-GCUCUCAGACUACGAUAUUNN-3′ SEQ ID NO: 4 3′-NNCGAGAGUCUGAUGCUAUAA-5′.SEQ ID NO: 5

Each “N” residue can be any nucleotide (A, C, G, U, T) or modifiednucleotide. The 3′ end can have a number of “N” residues between andincluding 1, 2, 3, 4, 5, and 6. The “N” residues on either strand can bethe same residue (e.g., UU, AA, CC, GG, or TT) or they can be different(e.g., AC, AG, AU, CA, CG, CU, GA, GC, GU, UA, UC, or UG). The 3′overhangs can be the same or they can be different. In one embodiment,both strands have a 3′UU overhang.

An example of an siRNA of the invention for targeting a correspondingmRNA sequence of SEQ ID NO:3 and having 21-nucleotide strands and a 3′UUoverhang on each strand is:

5′-GCUCUCAGACUACGAUAUUUU-3′ SEQ ID NO: 6 3′-UUCGAGAGUCUGAUGCUAUAA-5′.SEQ ID NO: 7

The interfering RNA may also have a 5′ overhang of nucleotides or it mayhave blunt ends. An siRNA of the invention for targeting a correspondingmRNA sequence of SEQ ID NO:3 and having 19-nucleotide strands and bluntends is:

5′-GCUCUCAGACUACGAUAUU-3′ SEQ ID NO: 8 3′-CGAGAGUCUGAUGCUAUAA-5′.SEQ ID NO: 9

The strands of a double-stranded interfering RNA (e.g., an siRNA) may beconnected to form a hairpin or stem-loop structure (e.g., an shRNA). AnshRNA of the invention targeting a corresponding mRNA sequence of SEQ IDNO:3 and having a 19 bp double-stranded stem region and a 3′UU overhangis:

N is a nucleotide A, T, C, G, U, or a modified form known by one ofordinary skill in the art. The number of nucleotides N in the loop is anumber between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9,or 9 to 11, or the number of nucleotides N is 9. Some of the nucleotidesin the loop can be involved in base-pair interactions with othernucleotides in the loop. Examples of oligonucleotide sequences that canbe used to form the loop include 5′-UUCAAGAGA-3′ (Brummelkamp, T. R. etal. (2002) Science 296: 550) and 5′-UUUGUGUAG-3′ (Castanotto, D. et al.(2002) RNA 8:1454). It will be recognized by one of skill in the artthat the resulting single chain oligonucleotide forms a stem-loop orhairpin structure comprising a double-stranded region capable ofinteracting with the RNAi machinery.

The siRNA target sequence identified above can be extended at the 3′ endto facilitate the design of dicer-substrate 27-mer duplexes. Extensionof the 19-nucleotide DNA target sequence (SEQ ID NO:3) identified in theTACE DNA sequence (SEQ ID NO:1) by 6 nucleotides yields a 25-nucleotideDNA target sequence present at nucleotides 297 to 321 of SEQ ID NO:1:

5′-GCTCTCAGACTACGATATTCTCTCT-3′. SEQ ID NO: 11

An example of a dicer-substrate 27-mer duplex of the invention fortargeting a corresponding mRNA sequence of SEQ ID NO:11 is:

5′-GCUCUCAGACUACGAUAUUCUCUCU-3′ SEQ ID NO: 123′-UUCGAGAGUCUGAUGCUAUAAGAGAGA-5′. SEQ ID NO: 13

The two nucleotides at the 3′ end of the sense strand (i.e., the CUnucleotides of SEQ ID NO:12) may be deoxynucleotides for enhancedprocessing. Design of dicer-substrate 27-mer duplexes from 19-21nucleotide target sequences, such as provided herein, is furtherdiscussed by the Integrated DNA Technologies (IDT) website and by Kim,D. -H. et al., (February, 2005) Nature Biotechnology 23:2; 222-226.

The target RNA cleavage reaction guided by siRNAs and other forms ofinterfering RNA is highly sequence specific. For example, in general, ansiRNA molecule contains a sense nucleotide strand identical in sequenceto a portion of the target mRNA and an antisense nucleotide strandexactly complementary to a portion of the target for inhibition of mRNAexpression. However, 100% sequence complementarity between the antisensesiRNA strand and the target mRNA, or between the antisense siRNA strandand the sense siRNA strand, is not required to practice the presentinvention, so long as the interfering RNA can recognize the target mRNAand silence expression of the TACE or TNFR1 gene. Thus, for example, theinvention allows for sequence variations between the antisense strandand the target mRNA and between the antisense strand and the sensestrand, including nucleotide substitutions that do not affect activityof the interfering RNA molecule, as well as variations that might beexpected due to genetic mutation, strain polymorphism, or evolutionarydivergence, wherein the variations do not preclude recognition of theantisense strand to the target mRNA.

In one embodiment of the invention, interfering RNA of the invention hasa sense strand and an antisense strand, and the sense and antisensestrands comprise a region of at least near-perfect contiguouscomplementarity of at least 19 nucleotides. In another embodiment of theinvention, an interfering RNA of the invention has a sense strand and anantisense strand, and the antisense strand comprises a region of atleast near-perfect contiguous complementarity of at least 19 nucleotidesto a target sequence of TACE or TNFR1 mRNA, and the sense strandcomprises a region of at least near-perfect contiguous identity of atleast 19 nucleotides with a target sequence of TACE or TNFR1 mRNA,respectively. In a further embodiment of the invention, the interferingRNA comprises a region of at least 13, 14, 15, 16, 17, or 18 contiguousnucleotides having percentages of sequence complementarity to or, havingpercentages of sequence identity with, the penultimate 13, 14, 15, 16,17, or 18 nucleotides, respectively, of the 3′ end of the correspondingtarget sequence within an mRNA. The length of each strand of theinterfering RNA comprises about 19 to about 49 nucleotides, and maycomprise a length of about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, or 49 nucleotides.

In certain embodiments, the antisense strand of an interfering RNA ofthe invention has at least near-perfect contiguous complementarity of atleast 19 nucleotides with the target mRNA. “Near-perfect,” as usedherein, means the antisense strand of the siRNA is “substantiallycomplementary to,” and the sense strand of the siRNA is “substantiallyidentical to” at least a portion of the target mRNA. “Identity,” asknown by one of ordinary skill in the art, is the degree of sequencerelatedness between nucleotide sequences as determined by matching theorder and identity of nucleotides between the sequences. In oneembodiment, the antisense strand of an siRNA having 80% and between 80%up to 100% complementarity, for example, 85%, 90% or 95%complementarity, to the target mRNA sequence are considered near-perfectcomplementarity and may be used in the present invention. “Perfect”contiguous complementarity is standard Watson-Crick base pairing ofadjacent base pairs. “At least near-perfect” contiguous complementarityincludes “perfect” complementarity as used herein. Computer methods fordetermining identity or complementarity are designed to identify thegreatest degree of matching of nucleotide sequences, for example, BLASTN(Altschul, S. F., et al. (1990) J. Mol. Biol. 215:403-410).

The term “percent identity” describes the percentage of contiguousnucleotides in a first nucleic acid molecule that is the same as in aset of contiguous nucleotides of the same length in a second nucleicacid molecule. The term “percent complementarity” describes thepercentage of contiguous nucleotides in a first nucleic acid moleculethat can base pair in the Watson-Crick sense with a set of contiguousnucleotides in a second nucleic acid molecule.

The relationship between a target mRNA and one strand of an siRNA (thesense strand) is that of identity. The sense strand of an siRNA is alsocalled a passenger strand, if present. The relationship between a targetmRNA and the other strand of an siRNA (the antisense strand) is that ofcomplementarity. The antisense strand of an siRNA is also called a guidestrand.

There may be a region or regions of the antisense siRNA strand that is(are) not complementary to a portion of SEQ ID NO: 1 or a portion of SEQID NO: 2. Non-complementary regions may be at the 3′, 5′ or both ends ofa complementary region or between two complementary regions. A regioncan be one or more bases.

The sense and antisense strands in an interfering RNA molecule can alsocomprise nucleotides that do not form base pairs with the other strand.For example, one or both strands can comprise additional nucleotides ornucleotides that do not pair with a nucleotide in that position on theother strand, such that a bulge or a mismatch is formed when the strandsare hybridized. Thus, an interfering RNA molecule of the invention cancomprise sense and antisense strands having mismatches, G-U wobbles, orbulges. Mismatches, G-U wobbles, and bulges can also occur between theantisense strand and its target (see, for example, Saxena et al., 2003,J. Biol. Chem. 278:44312-9).

One or both of the strands of double-stranded interfering RNA may have a3′ overhang of from 1 to 6 nucleotides, which may be ribonucleotides ordeoxyribonucleotides or a mixture thereof. The nucleotides of theoverhang are not base-paired. In one embodiment of the invention, theinterfering RNA comprises a 3′ overhang of TT or UU. In anotherembodiment of the invention, the interfering RNA comprises at least oneblunt end. The termini usually have a 5′ phosphate group or a 3′hydroxyl group. In other embodiments, the antisense strand has a 5′phosphate group, and the sense strand has a 5′ hydroxyl group. In stillother embodiments, the termini are further modified by covalent additionof other molecules or functional groups.

The sense and antisense strands of the double-stranded siRNA may be in aduplex formation of two single strands as described above or may be asingle-stranded molecule where the regions of complementarity arebase-paired and are covalently linked by a linker molecule to form ahairpin loop when the regions are hybridized to each other. It isbelieved that the hairpin is cleaved intracellularly by a protein termeddicer to form an interfering RNA of two individual base-paired RNAmolecules. A linker molecule can also be designed to comprise arestriction site that can be cleaved in vivo or in vitro by a particularnuclease.

In one embodiment, the invention provides an interfering RNA moleculethat comprises a region of at least 13 contiguous nucleotides having atleast 90% sequence complementarity to, or at least 90% sequence identitywith, the penultimate 13 nucleotides of the 3′ end of an mRNAcorresponding to a DNA target, which allows a one nucleotidesubstitution within the region. Two nucleotide substitutions (i.e.,11/13=85% identity/complementarity) are not included in such a phrase.In another embodiment, the invention provides an interfering RNAmolecule that comprises a region of at least 14 contiguous nucleotideshaving at least 85% sequence complementarity to, or at least 85%sequence identity with, the penultimate 14 nucleotides of the 3′ end ofan mRNA corresponding to a DNA target. Two nucleotide substitutions(i.e., 12/14=86% identity/complementarity) are included in such aphrase. In a further embodiment, the invention provides an interferingRNA molecule that comprises a region of at least 15, 16, 17, or 18contiguous nucleotides having at least 80% sequence complementarity to,or at least 80% sequence identity with, the penultimate 14 nucleotidesof the 3′ end of an mRNA corresponding to a DNA target. Three nucleotidesubstitutions are included in such a phrase.

The penultimate base in a nucleic acid sequence that is written in a 5′to 3′ direction is the next to the last base, i.e., the base next to the3′ base. The penultimate 13 bases of a nucleic acid sequence written ina 5′ to 3′ direction are the last 13 bases of a sequence next to the 3′base and not including the 3′ base. Similarly, the penultimate 14, 15,16, 17, or 18 bases of a nucleic acid sequence written in a 5′ to 3′direction are the last 14, 15, 16, 17, or 18 bases of a sequence,respectively, next to the 3′ base and not including the 3′ base.

Interfering RNAs may be generated exogenously by chemical synthesis, byin vitro transcription, or by cleavage of longer double-stranded RNAwith dicer or another appropriate nuclease with similar activity.Chemically synthesized interfering RNAs, produced from protectedribonucleoside phosphoramidites using a conventional DNA/RNAsynthesizer, may be obtained from commercial suppliers such as AmbionInc. (Austin, Tex.), Invitrogen (Carlsbad, Calif.), or Dharmacon(Lafayette, Colo.). Interfering RNAs can be purified by extraction witha solvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof, for example. Alternatively, interfering RNA may beused with little if any purification to avoid losses due to sampleprocessing.

When interfering RNAs are produced by chemical synthesis,phosphorylation at the 5′ position of the nucleotide at the 5′ end ofone or both strands (when present) can enhance siRNA efficacy andspecificity of the bound RISC complex, but is not required sincephosphorylation can occur intracellularly.

Interfering RNAs can also be expressed endogenously from plasmid orviral expression vectors or from minimal expression cassettes, forexample, PCR generated fragments comprising one or more promoters and anappropriate template or templates for the interfering RNA. Examples ofcommercially available plasmid-based expression vectors for shRNAinclude members of the pSilencer series (Ambion, Austin, Tex.) andpCpG-siRNA (InvivoGen, San Diego, Calif.). Viral vectors for expressionof interfering RNA may be derived from a variety of viruses includingadenovirus, adeno-associated virus, lentivirus (e.g., HIV, FIV, andEIAV), and herpes virus. Examples of commercially available viralvectors for shRNA expression include pSilencer adeno (Ambion, Austin,Tex.) and pLenti6/BLOCK-iT™-DEST (Invitrogen, Carlsbad, Calif.).Selection of viral vectors, methods for expressing the interfering RNAfrom the vector and methods of delivering the viral vector are withinthe ordinary skill of one in the art. Examples of kits for production ofPCR-generated shRNA expression cassettes include Silencer Express(Ambion, Austin, Tex.) and siXpress (Mirus, Madison, Wis.).

In certain embodiments, a first interfering RNA may be administered viain vivo expression from a first expression vector capable of expressingthe first interfering RNA and a second interfering RNA may beadministered via in vivo expression from a second expression vectorcapable of expressing the second interfering RNA, or both interferingRNAs may be administered via in vivo expression from a single expressionvector capable of expressing both interfering RNAs. Additionalinterfering RNAs can be administered in a like manner (i.e. via separateexpression vectors or via a single expression vector capable ofexpressing multiple interfering RNAs).

Interfering RNAs may be expressed from a variety of eukaryotic promotersknown to those of ordinary skill in the art, including pol IIIpromoters, such as the U6 or H1 promoters, or pol II promoters, such asthe cytomegalovirus promoter. Those of skill in the art will recognizethat these promoters can also be adapted to allow inducible expressionof the interfering RNA.

In certain embodiments of the present invention, an antisense strand ofan interfering RNA hybridizes with an mRNA in vivo as part of the RISCcomplex.

“Hybridization” refers to a process in which single-stranded nucleicacids with complementary or near-complementary base sequences interactto form hydrogen-bonded complexes called hybrids. Hybridizationreactions are sensitive and selective. In vitro, the specificity ofhybridization (i.e., stringency) is controlled by the concentrations ofsalt or formamide in prehybridization and hybridization solutions, forexample, and by the hybridization temperature; such procedures are wellknown in the art. In particular, stringency is increased by reducing theconcentration of salt, increasing the concentration of formamide, orraising the hybridization temperature.

For example, high stringency conditions could occur at about 50%formamide at 37° C. to 42° C. Reduced stringency conditions could occurat about 35% to 25% formamide at 30° C. to 35° C. Examples of stringencyconditions for hybridization are provided in Sambrook, J., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. Further examples of stringenthybridization conditions include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing, orhybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamidefollowed by washing at 70° C. in 0.3×SSC, or hybridization at 70° C. in4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in1×SSC. The temperature for hybridization is about 5-10° C. less than themelting temperature (T_(m)) of the hybrid where T_(m) is determined forhybrids between 19 and 49 base pairs in length using the followingcalculation: T_(m)° C.=81.5+16.6(log₁₀[Na+])+0.41 (% G+C)−(600/N) whereN is the number of bases in the hybrid, and [Na+] is the concentrationof sodium ions in the hybridization buffer.

The above-described in vitro hybridization assay provides a method ofpredicting whether binding between a candidate siRNA and a target willhave specificity. However, in the context of the RISC complex, specificcleavage of a target can also occur with an antisense strand that doesnot demonstrate high stringency for hybridization in vitro.

Interfering RNAs may differ from naturally-occurring RNA by theaddition, deletion, substitution or modification of one or morenucleotides. Non-nucleotide material may be bound to the interferingRNA, either at the 5′ end, the 3′ end, or internally. Such modificationsare commonly designed to increase the nuclease resistance of theinterfering RNAs, to improve cellular uptake, to enhance cellulartargeting, to assist in tracing the interfering RNA, to further improvestability, or to reduce the potential for activation of the interferonpathway. For example, interfering RNAs may comprise a purine nucleotideat the ends of overhangs. Conjugation of cholesterol to the 3′ end ofthe sense strand of an siRNA molecule by means of a pyrrolidine linker,for example, also provides stability to an siRNA.

Further modifications include a 3′ terminal biotin molecule, a peptideknown to have cell-penetrating properties, a nanoparticle, apeptidomimetic, a fluorescent dye, or a dendrimer, for example.

Nucleotides may be modified on their base portion, on their sugarportion, or on the phosphate portion of the molecule and function inembodiments of the present invention.

Modifications include substitutions with alkyl, alkoxy, amino, deaza,halo, hydroxyl, thiol groups, or a combination thereof, for example.Nucleotides may be substituted with analogs with greater stability suchas replacing a ribonucleotide with a deoxyribonucleotide, or havingsugar modifications such as 2′ OH groups replaced by 2′ amino groups, 2′O-methyl groups, 2′ methoxyethyl groups, or a 2′-O, 4′-C methylenebridge, for example. Examples of a purine or pyrimidine analog ofnucleotides include a xanthine, a hypoxanthine, an azapurine, amethylthioadenine, 7-deaza-adenosine and O- and N-modified nucleotides.The phosphate group of the nucleotide may be modified by substitutingone or more of the oxygens of the phosphate group with nitrogen or withsulfur (phosphorothioates). Modifications are useful, for example, toenhance function, to improve stability or permeability, or to directlocalization or targeting.

In certain embodiments, an interfering molecule of the inventioncomprises at least one of the modifications as described above.

In certain embodiments, the invention provides pharmaceuticalcompositions (also referred to herein as “compositions”) comprising aninterfering RNA molecule of the invention. Pharmaceutical compositionsare formulations that comprise interfering RNAs, or salts thereof, ofthe invention up to 99% by weight mixed with a physiologicallyacceptable carrier medium, including those described infra, and such aswater, buffer, saline, glycine, hyaluronic acid, mannitol, and the like.

Interfering RNAs of the present invention are administered as solutions,suspensions, or emulsions. The following are examples of pharmaceuticalcomposition formulations that may be used in the methods of theinvention.

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50;  0.5-10.0Hydroxypropylmethylcellulose 0.5 Sodium chloride 0.8 BenzalkoniumChloride 0.01 EDTA 0.01 NaOH/HCl qs pH 7.4 Purified water (RNase-free)qs 100 mL

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50;  0.5-10.0Phosphate Buffered Saline 1.0 Benzalkonium Chloride 0.01 Polysorbate 800.5 Purified water (RNase-free) q.s. to 100%

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50;  0.5-10.0Monobasic sodium phosphate 0.05 Dibasic sodium phosphate 0.15(anhydrous) Sodium chloride 0.75 Disodium EDTA 0.05 Cremophor EL 0.1Benzalkonium chloride 0.01 HCl and/or NaOH pH 7.3-7.4 Purified water(RNase-free) q.s. to 100%

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50;  0.5-10.0Phosphate Buffered Saline 1.0 Hydroxypropyl-β-cyclodextrin 4.0 Purifiedwater (RNase-free) q.s. to 100%

As used herein the term “effective amount” refers to the amount ofinterfering RNA or a pharmaceutical composition comprising aninterfering RNA determined to produce a therapeutic response in amammal. Such therapeutically effective amounts are readily ascertainedby one of ordinary skill in the art and using methods as describedherein.

Generally, an effective amount of the interfering RNAs of the inventionresults in an extracellular concentration at the surface of the targetcell of from 100 pM to 1 μM, or from 1 nM to 100 nM, or from 5 nM toabout 50 nM, or to about 25 nM. The dose required to achieve this localconcentration will vary depending on a number of factors including thedelivery method, the site of delivery, the number of cell layers betweenthe delivery site and the target cell or tissue, whether delivery islocal or systemic, etc. The concentration at the delivery site may beconsiderably higher than it is at the surface of the target cell ortissue. Topical compositions can be delivered to the surface of thetarget organ, such as the eye, one to four times per day, or on anextended delivery schedule such as daily, weekly, bi-weekly, monthly, orlonger, according to the routine discretion of a skilled clinician. ThepH of the formulation is about pH 4.0 to about pH 9.0, or about pH 4.5to about pH 7.4.

An effective amount of a formulation may depend on factors such as theage, race, and sex of the subject, the rate of target genetranscript/protein turnover, the interfering RNA potency, and theinterfering RNA stability, for example. In one embodiment, theinterfering RNA is delivered topically to a target organ and reaches theTACE or TNFR1 mRNA-containing tissue such as the trabecular meshwork,retina or optic nerve head at a therapeutic dose thereby amelioratingTNFα-associated disease process.

Therapeutic treatment of patients with interfering RNAs directed againstTACE or TNFR1 mRNA is expected to be beneficial over small moleculetreatments by increasing the duration of action, thereby allowing lessfrequent dosing and greater patient compliance, and by increasing targetspecificity, thereby reducing side effects.

An “acceptable carrier” as used herein refers to those carriers thatcause at most, little to no ocular irritation, provide suitablepreservation if needed, and deliver one or more interfering RNAs of thepresent invention in a homogenous dosage. An acceptable carrier foradministration of interfering RNA of embodiments of the presentinvention include the cationic lipid-based transfection reagentsTransIT®-TKO (Mirus Corporation, Madison, Wis.), LIPOFECTIN®,Lipofectamine, OLIGOFECTAMINE™ (Invitrogen, Carlsbad, Calif.), orDHARMAFECT™ (Dharmacon, Lafayette, Colo.); polycations such aspolyethyleneimine; cationic peptides such as Tat, polyarginine, orPenetratin (Antp peptide); nanoparticles; or liposomes. Liposomes areformed from standard vesicle-forming lipids and a sterol, such ascholesterol, and may include a targeting molecule such as a monoclonalantibody having binding affinity for cell surface antigens, for example.Further, the liposomes may be PEGylated liposomes.

The interfering RNAs may be delivered in solution, in suspension, or inbioerodible or non-bioerodible delivery devices. The interfering RNAscan be delivered alone or as components of defined, covalent conjugates.The interfering RNAs can also be complexed with cationic lipids,cationic peptides, or cationic polymers; complexed with proteins, fusionproteins, or protein domains with nucleic acid binding properties (e.g.,protamine); or encapsulated in nanoparticles or liposomes. Tissue- orcell-specific delivery can be accomplished by the inclusion of anappropriate targeting moiety such as an antibody or antibody fragment.

Interfering RNA may be delivered via aerosol, buccal, dermal,intradermal, inhaling, intramuscular, intranasal, intraocular,intrapulmonary, intravenous, intraperitoneal, nasal, ocular, oral, otic,parenteral, patch, subcutaneous, sublingual, topical, or transdermaladministration, for example.

In certain embodiments, treatment of ocular disorders with interferingRNA molecules is accomplished by administration of an interfering RNAmolecule directly to the eye. Local administration to the eye isadvantageous for a number or reasons, including: the dose can be smallerthan for systemic delivery, and there is less chance of the moleculessilencing the gene target in tissues other than in the eye.

A number of studies have shown successful and effective in vivo deliveryof interfering RNA molecules to the eye. For example, Kim et al.demonstrated that subconjunctival injection and systemic delivery ofsiRNAs targeting VEGF pathway genes inhibited angiogenesis in a mouseeye (Kim et al., 2004, Am. J. Pathol. 165:2177-2185). In addition,studies have shown that siRNA delivered to the vitreous cavity candiffuse throughout the eye, and is detectable up to five days afterinjection (Campochiaro, 2006, Gene Therapy 13:559-562).

Interfering RNA may be delivered directly to the eye by ocular tissueinjection such as periocular, conjunctival, subtenon, intracameral,intravitreal, intraocular, subretinal, subconjunctival, retrobulbar, orintracanalicular injections; by direct application to the eye using acatheter or other placement device such as a retinal pellet, intraocularinsert, suppository or an implant comprising a porous, non-porous, orgelatinous material; by topical ocular drops or ointments; or by a slowrelease device in the cul-de-sac or implanted adjacent to the sclera(transscleral) or in the sclera (intrascleral) or within the eye.Intracameral injection may be through the cornea into the anteriorchamber to allow the agent to reach the trabecular meshwork.Intracanalicular injection may be into the venous collector channelsdraining Schlemm's canal or into Schlemm's canal.

For ophthalmic delivery, an interfering RNA may be combined withophthalmologically acceptable preservatives, co-solvents, surfactants,viscosity enhancers, penetration enhancers, buffers, sodium chloride, orwater to form an aqueous, sterile ophthalmic suspension or solution.Solution formulations may be prepared by dissolving the interfering RNAin a physiologically acceptable isotonic aqueous buffer. Further, thesolution may include an acceptable surfactant to assist in dissolvingthe interfering RNA. Viscosity building agents, such as hydroxymethylcellulose, hydroxyethyl cellulose, methylcellulose,polyvinylpyrrolidone, or the like may be added to the compositions ofthe present invention to improve the retention of the compound.

In order to prepare a sterile ophthalmic ointment formulation, theinterfering RNA is combined with a preservative in an appropriatevehicle, such as mineral oil, liquid lanolin, or white petrolatum.Sterile ophthalmic gel formulations may be prepared by suspending theinterfering RNA in a hydrophilic base prepared from the combination of,for example, CARBOPOL®-940 (BF Goodrich, Charlotte, N.C.), or the like,according to methods known in the art. VISCOAT® (Alcon Laboratories,Inc., Fort Worth, Tex.) may be used for intraocular injection, forexample. Other compositions of the present invention may containpenetration enhancing agents such as cremephor and TWEEN® 80(polyoxyethylene sorbitan monolaureate, Sigma Aldrich, St. Louis, Mo.),in the event the interfering RNA is less penetrating in the eye.

In certain embodiments, the invention also provides a kit that includesreagents for attenuating the expression of an mRNA as cited herein in acell. The kit contains an siRNA or an shRNA expression vector. ForsiRNAs and non-viral shRNA expression vectors the kit also contains atransfection reagent or other suitable delivery vehicle. For viral shRNAexpression vectors, the kit may contain the viral vector and/or thenecessary components for viral vector production (e.g., a packaging cellline as well as a vector comprising the viral vector template andadditional helper vectors for packaging). The kit may also containpositive and negative control siRNAs or shRNA expression vectors (e.g.,a non-targeting control siRNA or an siRNA that targets an unrelatedmRNA). The kit also may contain reagents for assessing knockdown of theintended target gene (e.g., primers and probes for quantitative PCR todetect the target mRNA and/or antibodies against the correspondingprotein for western blots). Alternatively, the kit may comprise an siRNAsequence or an shRNA sequence and the instructions and materialsnecessary to generate the siRNA by in vitro transcription or toconstruct an shRNA expression vector.

A pharmaceutical combination in kit form is further provided thatincludes, in packaged combination, a carrier means adapted to receive acontainer means in close confinement therewith and a first containermeans including an interfering RNA composition and an acceptablecarrier. Such kits can further include, if desired, one or more ofvarious conventional pharmaceutical kit components, such as, forexample, containers with one or more pharmaceutically acceptablecarriers, additional containers, etc., as will be readily apparent tothose skilled in the art. Printed instructions, either as inserts or aslabels, indicating quantities of the components to be administered,guidelines for administration, and/or guidelines for mixing thecomponents, can also be included in the kit.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated by reference.

Those of skill in the art, in light of the present disclosure, willappreciate that obvious modifications of the embodiments disclosedherein can be made without departing from the spirit and scope of theinvention. All of the embodiments disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. The full scope of the invention is set out in the disclosureand equivalent embodiments thereof. The specification should not beconstrued to unduly narrow the full scope of protection to which thepresent invention is entitled.

While a particular embodiment of the invention has been shown anddescribed, numerous variations and alternate embodiments will occur tothose skilled in the art. Accordingly, the invention may be embodied inother specific forms without departing from its spirit or essentialcharacteristics. The described embodiments are to be considered in allrespects only as illustrative and not restrictive. The scope of theinvention is, therefore, indicated by the appended claims rather than bythe foregoing description. All changes to the claims that come withinthe meaning and range of equivalency of the claims are to be embracedwithin their scope. Further, all published documents, patents, andapplications mentioned herein are hereby incorporated by reference, asif presented in their entirety.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved, are provided for illustrative purposes only and are not to beconstrued as limiting the invention.

Example 1 Interfering RNA for Specifically Silencing TNFR1 in GTM-3Cells

The present study examines the ability of TNFR1 interfering RNA to knockdown the levels of endogenous TNFR1 protein expression in cultured GTM-3cells.

Transfection of GTM-3 cells (Pang, I. H. et al., 1994. Curr. Eye Res.13:51-63) was accomplished using standard in vitro concentrations(0.1-10 nM) of TNFR1 siRNAs, siCONTROL RISC-free siRNA, or siCONTROLNon-targeting siRNA #2 (NTC2) and DHARMAFECT® #1 transfection reagent(Dharmacon, Lafayette, Colo.). All siRNAs were dissolved in 1× siRNAbuffer, an aqueous solution of 20 mM KCl, 6 mM HEPES (pH 7.5), 0.2 mMMgCl₂. Control samples included a buffer control in which the volume ofsiRNA was replaced with an equal volume of 1× siRNA buffer (-siRNA).Western blots using an anti-TNFR1 antibody (Santa Cruz Biotechnology,Santa Cruz, Calif.) were performed to assess TNFR1 protein expression.The TNFR1 siRNAs are double-stranded interfering RNAs having specificityfor the following targets: siTNFR1 #1 targets the sequenceCAAAGGAACCUACUUGUAC (SEQ ID NO: 202); siTNFR1 #2 targets the sequenceGAGCUUGAAGGAACUACUA (SEQ ID NO: 203); siTNFR1 #3 targets the sequenceCACAGAGCCUAGACACUGA (SEQ ID NO: 204); siTNFR1 #4 targets the sequenceUCCAAGCUCUACUCCAUUG (SEQ ID NO: 205). As shown by the data in FIG. 1,siTNFR1 #1, siTNFR1 #2, and siTNFR1 #3 siRNAs reduced TNFR1 proteinexpression significantly at the 10 nM and 1 nM concentrations relativeto the control siRNAs, but exhibited reduced efficacy at 0.1 nM. ThesiTNFR1 #2 and siTNFR1 #3 siRNAs were particularly effective. ThesiTNFR1 #4 siRNA also showed a concentration dependent reduction inTNFR1 protein expression as expected.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

What is claimed is:
 1. A method of treating a TNFα-related oculardisorder in a patient in need thereof, comprising administering to thepatient a composition comprising an effective amount of a doublestranded interfering RNA having a length of 19 to 49 nucleotides, and apharmaceutically acceptable carrier, the interfering RNA comprising asense nucleotide strand, and an antisense nucleotide strand, wherein theantisense strand hybridizes under physiological conditions to a portionof mRNA corresponding to SEQ ID NO:2 comprising nucleotide 1573, andwherein the TNFα-related ocular disorder is ocular hypertension orglaucoma.
 2. The method of claim 1, wherein the interfering RNA moleculeis double stranded and each strand is independently about 19 to about 27nucleotides in length.
 3. The method of claim 2, wherein each strand isindependently about 19 nucleotides to about 25 nucleotides in length. 4.The method of claim 2, wherein each strand is independently about 19nucleotides to about 21 nucleotides in length.
 5. The method of claim 2,wherein the sense and antisense strands are connected by a linker toform a shRNA that can attenuate expression of TNFR1 mRNA in a patient.6. The method of claim 2, wherein the interfering RNA molecule has bluntends.
 7. The method of claim 2, wherein at least one strand of theinterfering RNA molecule comprises a 3′ overhang.
 8. The method of claim7, wherein the 3′ overhang comprises about 1 to about 6 nucleotides. 9.The method of claim 8, wherein the 3′ overhang comprises 2 nucleotides.10. The method of claim 1, wherein the interfering RNA molecule isadministered via in vivo expression from an expression vector capable ofexpressing the interfering RNA molecule.
 11. The method of claim 1,wherein the interfering RNA molecule recognizes a portion of TNFR1 mRNAthat corresponds to SEQ ID NO:
 154. 12. The method of claim 1, whereinthe interfering RNA molecule comprises at least one modification. 13.The composition of claim 1, wherein the interfering RNA molecule is ashRNA, a siRNA, or a miRNA.
 14. The method of claim 1, wherein theinterfering RNA molecule is administered via a topical, intravitreal,transcleral, periocular, conjunctival, subtenon, intracameral,subretinal, subconjunctival, retrobulbar, or intracanalicular route.